Neuropeptide regulation of non-redundant ILC2 responses at barrier surfaces

doi:10.1038/s41586-022-05297-6

. 2022 Nov;611(7937):787-793.

doi: 10.1038/s41586-022-05297-6. Epub 2022 Nov 2.

Neuropeptide regulation of non-redundant ILC2 responses at barrier surfaces

Amy M Tsou^#^{1

2}, Hiroshi Yano^#^{1

3

4}, Christopher N Parkhurst^{1

3

4}, Tanel Mahlakõiv¹, Coco Chu¹, Wen Zhang^{1

3

4}, Zhengxiang He⁵, Katja J Jarick⁶, Connie Zhong¹, Gregory G Putzel¹, Mai Hatazaki¹; JRI IBD Live Cell Bank Consortium; Ivo C Lorenz⁷, David Andrew⁷, Paul Balderes⁷, Christoph S N Klose⁶, Sergio A Lira⁵, David Artis^{8

9

10

11}

Collaborators, Affiliations

Collaborators

JRI IBD Live Cell Bank Consortium:
Randy Longman, Gregory Sonnenberg, Ellen Scherl, Dana Lukin, Robert Battat, Robbyn Sockolow, Thomas Ciecierega, Aliza Solomon, Elaine Barfield, Kimberley Chien, Johanna Ferreira, Jasmin Williams, Shaira Khan, Peik Sean Chong, Samah Mozumder, Lance Chou, Wenqing Zhou, Anees Ahmed, Ann M Joseph

Affiliations

¹ Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, New York, NY, USA.
² Division of Pediatric Gastroenterology, Hepatology and Nutrition, Weill Cornell Medical College, New York, NY, USA.
³ Joan and Sanford I. Weill Department of Medicine, Division of Gastroenterology and Hepatology, Weill Cornell Medicine, Cornell University, New York, NY, USA.
⁴ Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA.
⁵ Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
⁶ Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Microbiology, Infectious Diseases and Immunology, Berlin, Germany.
⁷ Tri-Institutional Therapeutics Discovery Institute, New York, NY, USA.
⁸ Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, New York, NY, USA. dartis@med.cornell.edu.
⁹ Joan and Sanford I. Weill Department of Medicine, Division of Gastroenterology and Hepatology, Weill Cornell Medicine, Cornell University, New York, NY, USA. dartis@med.cornell.edu.
¹⁰ Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA. dartis@med.cornell.edu.
¹¹ Friedman Center for Nutrition and Inflammation, Joan and Sanford I. Weill Department of Medicine, Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA. dartis@med.cornell.edu.

^# Contributed equally.

PMID: 36323781
PMCID: PMC10225046
DOI: 10.1038/s41586-022-05297-6

Neuropeptide regulation of non-redundant ILC2 responses at barrier surfaces

Amy M Tsou et al. Nature. 2022 Nov.

. 2022 Nov;611(7937):787-793.

doi: 10.1038/s41586-022-05297-6. Epub 2022 Nov 2.

Authors

Collaborators

JRI IBD Live Cell Bank Consortium:
Randy Longman, Gregory Sonnenberg, Ellen Scherl, Dana Lukin, Robert Battat, Robbyn Sockolow, Thomas Ciecierega, Aliza Solomon, Elaine Barfield, Kimberley Chien, Johanna Ferreira, Jasmin Williams, Shaira Khan, Peik Sean Chong, Samah Mozumder, Lance Chou, Wenqing Zhou, Anees Ahmed, Ann M Joseph

Affiliations

¹ Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, New York, NY, USA.
² Division of Pediatric Gastroenterology, Hepatology and Nutrition, Weill Cornell Medical College, New York, NY, USA.
³ Joan and Sanford I. Weill Department of Medicine, Division of Gastroenterology and Hepatology, Weill Cornell Medicine, Cornell University, New York, NY, USA.
⁴ Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA.
⁵ Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA.
⁶ Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Microbiology, Infectious Diseases and Immunology, Berlin, Germany.
⁷ Tri-Institutional Therapeutics Discovery Institute, New York, NY, USA.
⁸ Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, New York, NY, USA. dartis@med.cornell.edu.
⁹ Joan and Sanford I. Weill Department of Medicine, Division of Gastroenterology and Hepatology, Weill Cornell Medicine, Cornell University, New York, NY, USA. dartis@med.cornell.edu.
¹⁰ Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA. dartis@med.cornell.edu.
¹¹ Friedman Center for Nutrition and Inflammation, Joan and Sanford I. Weill Department of Medicine, Department of Microbiology and Immunology, Weill Cornell Medicine, Cornell University, New York, NY, USA. dartis@med.cornell.edu.

^# Contributed equally.

PMID: 36323781
PMCID: PMC10225046
DOI: 10.1038/s41586-022-05297-6

Abstract

Emerging studies indicate that cooperation between neurons and immune cells regulates antimicrobial immunity, inflammation and tissue homeostasis. For example, a neuronal rheostat provides excitatory or inhibitory signals that control the functions of tissue-resident group 2 innate lymphoid cells (ILC2s) at mucosal barrier surfaces^1-4. ILC2s express NMUR1, a receptor for neuromedin U (NMU), which is a prominent cholinergic neuropeptide that promotes ILC2 responses^5-7. However, many functions of ILC2s are shared with adaptive lymphocytes, including the production of type 2 cytokines^8,9 and the release of tissue-protective amphiregulin (AREG)^10-12. Consequently, there is controversy regarding whether innate lymphoid cells and adaptive lymphocytes perform redundant or non-redundant functions^13-15. Here we generate a new genetic tool to target ILC2s for depletion or gene deletion in the presence of an intact adaptive immune system. Transgenic expression of iCre recombinase under the control of the mouse Nmur1 promoter enabled ILC2-specific deletion of AREG. This revealed that ILC2-derived AREG promotes non-redundant functions in the context of antiparasite immunity and tissue protection following intestinal damage and inflammation. Notably, NMU expression levels increased in inflamed intestinal tissues from both mice and humans, and NMU induced AREG production in mouse and human ILC2s. These results indicate that neuropeptide-mediated regulation of non-redundant functions of ILC2s is an evolutionarily conserved mechanism that integrates immunity and tissue protection.

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Conflict of interest statement

Competing interests

D.A. has contributed to scientific advisory boards at Pfizer, Takeda, FARE, and the KRF. The other authors declare no competing interests.

Figures

**Extended Data Figure 1 |. Nmur1-eGFP is highly expressed and enriched in ILC2s in lymphoid and non-lymphoid tissues.**
a, Percentages of surface NMUR1⁺ ILC2s isolated from the indicated tissues from *Nmur1*^+/+ WT mice (n=5 mice). b, Schematic of targeting construct for generating Nmur1^iCre-eGFP mice. **c-k**, Comprehensive analysis of Nmur1-eGFP expression in Nmur1^iCre-eGFP mice. Representative gating strategy for live CD45⁺ cells (c), innate lymphoid cell (ILC) subsets (d), adaptive lymphocytes (e), and myeloid cells and granulocytes (g). Representative overlaid histograms depicting Nmur1-eGFP expression in adaptive lymphocytes (f) and myeloid cells and granulocytes (h) from Nmur1^iCre-eGFP mice and Cre-negative littermate controls. Percentages of Nmur1-eGFP⁺ cells within the indicated immune cell subsets isolated from small intestines (i), mesenteric lymph nodes (MLN) (j), and spleens (k) of Nmur1^iCre-eGFP mice (n=3 mice). Data in a and **i-k** are representative of two independent experiments. Data are represented as means ± S.E.M.

**Extended Data Figure 2 |. ILC2-enriched eGFP expression and iCre-mediated induction of R26-RFP expression.**
a, Representative gating strategy. b, Representative histograms depicting Nmur1-eGFP expression in the indicated progenitor immune cell subsets in the bone marrow. c, Percentages of Nmur1-eGFP⁺ cells within the indicated progenitor cells isolated from Nmur1^iCre-eGFP mice (n=4 mice). d, Unbiased analysis of Nmur1-eGFP⁺ cells in the bone marrow by flow cytometry, depicting a representative of two independent experiments. e, Percentages of RFP⁺ ILC2s in indicated tissues from *R26-RFP*^Nmur1 mice (n=4 mice). **f-m,** Representative histograms showing iCre (RFP) expression in the indicated immune cell subsets isolated from the SI (f), colon (h), MLN (j), and spleen (l) of *R26-RFP*^Nmur1 mice (n=4 mice). Percentage of iCre (RFP)⁺ cells within the indicated immune cell subsets isolated from the SI (g), colon (i), MLN (k), and spleen (m). Data in **c, e, g, i, k, m** are pooled from of 2 independent experiments. Data are represented as means ± S.E.M. SI - small intestine, MLN - mesenteric lymph node, Neu – neutrophils, Eos – eosinophils, MΦ- macrophages, DC – dendritic cells.

**Extended Data Figure 3 |. Comparison of efficiency of ILC2-depletion and its impact on other immune cells in *R26-DTR*^Nmur1 and *R26-DTR*^Red5 mice.**
**a-c**, *R26-DTR* (n=11 mice) and *R26-DTR*^Nmur1 (n=7 mice) were treated with 2 daily injections of DT. The small intestine (SI), colon, mesenteric lymph nodes (MLN), and spleen were harvested for analysis after resting the mice for 2 days. Abundances of non-ILC2 innate lymphoid cell subsets (a), adaptive lymphocytes (b) or myeloid cells and granulocytes (c) within the indicated tissues. **d-g**, *R26-DTR* (n=7 mice) and *R26-DTR*^Red5 (n=6 mice) were treated with 2 daily injections of DT and rested for 2 days. Representative flow cytometry analysis of ILC2s in the colon, pre-gated on total ILCs (d) (Total ILCs gated as live CD45⁺Lin⁻CD90⁺CD127⁺ events. Lin: CD3ε, CD5, CD11b, CD11c, FcεRI, B220). Percentages of ILC2s out of total ILCs in the SI, colon, MLN, and spleen (e). Abundances of innate lymphoid cell subsets (f), adaptive lymphocytes (g). Data in **a-c** and **e-g** are pooled from two independent experiments. Two-way ANOVA with Šídák multiple comparisons test (**a-c, e-g**). P values are presented where appropriate. ns, not significant. Data are represented as means ± S.E.M.

**Extended Data Figure 4 |. Nmur1^iCre-eGFP-mediated AREG deletion does not impact AREG production in CD4⁺ T cells or intrinsic ILC2 homeostasis.**
**a-c**, Representative flow cytometry analysis of AREG and NMUR1 co-expression within ST2⁻ and ST2⁺ CD4⁺ T cells (a) and ILC2s (b) at steady state in the MLN of WT mice. Percentages of NMUR1⁺ cells within AREG-producing cells isolated from WT mice (n=3 mice) (c). d, Schematic of *Areg*^fl mouse generation using CRISPR/Cas9n. e, f, Representative flow cytometry analysis of ST2⁻ and ST2⁺ CD4⁺ T cells (e) and percentages of AREG-producing cells (f) in the MLN of *Areg*^fl (n=7 mice), *Areg*^ΔRed5 (n=5 mice), and *Areg*^ΔILC2 (n=5 mice). **g-l**, Small intestine and MLN from *Areg*^fl (n=4 mice) and *Areg*^ΔILC2 (n=5 mice) were analyzed at steady state for ILC2 abundance and their cytokine production capacity. Percentages of ILC2s within total ILCs in the small intestine (g) and MLN (i). Absolute cell numbers of ILC2s in the small intestine (h) and MLN (j). Percentages of IL-5⁺IL-13⁺ DP, total IL-5⁺ and total IL-13⁺ ILC2s isolated from the small intestine (k) and MLN (l). Data in **c, f, g-l** are representative of two independent experiments. One-way ANOVA with Tukey multiple comparisons test (**c, f**). Unpaired two-sided t-test (**g-l**). P values are presented where appropriate. ns, not significant. Data are represented as means ± S.E.M. MLN – mesenteric lymph node. DP – double positive.

**Extended Data Figure 5 |. Deletion of ILC2-derived AREG does not impact the priming of adaptive immune responses.**
**a-e**, Analysis of MLN isolated from naïve *Areg*^fl (n=3 mice) and *Trichuris*-infected *Areg*^fl (n=7 mice) and *Areg*^ΔILC2 (n=8 mice) at day 19 post-infection. Cecal patches (CP) were pooled as one sample when necessary due to cell yield for flow cytometry analysis (*Areg*^fl (n=2 pooled CP) and *Trichuris*-infected *Areg*^fl (n=3 pooled CP) and *Areg*^ΔILC2 (n=3 pooled CP)). Representative flow cytometry analysis of CD4⁺ T cell subsets (**a, b**) and percentages of Foxp3⁺ Tregs, T-bet⁺ Th1 and GATA3⁺ Th2 CD4⁺ T cells (c). Representative flow cytometry analysis of pathogen-specific cytokine production in MLN CD4⁺ T cells after stimulation with *Trichuris* antigen for 3 days *ex vivo* (d). Percentage of CD4⁺ T cells producing TNFα, IFNγ, IL-4, IL-5, and IL-13 (e). f, Detection of *Trichuris* antigen-specific IgG1 and IgG2c in serum from naïve *Areg*^fl (n=4 mice) and *Trichuris*-infected *Areg*^fl (n=12 mice) and *Areg*^ΔILC2 (n=13 mice) at day 19 post-infection by serial dilution ELISA. Data in c and e are pooled from two independent experiments. Data in f are pooled from three independent experiments. One-way ANOVA with Tukey multiple comparisons test (c, e). Two-way ANOVA with Šídák multiple comparisons test (f). ns, not significant. Data are represented as means ± S.E.M.

**Extended Data Figure 6 |. Bone marrow chimera mice recapitulated the phenotype of *Areg*^ΔILC2 mice during *Trichuris muris* infection.**
Irradiated CD45.1 WT mice received BM from *Areg*^fl or *Areg*^ΔILC2 donor mice. After 8 weeks, chimera mice were infected with 200 *Trichuris* eggs by oral gavage and analyzed 19 days post-infection. a, Experimental schematic. **b-d**, AREG deletion in ILC2 (b), ST2⁻ CD4⁺ T cells (c), and ST2⁺ CD4⁺ T cells (d) in *Trichuris-*infected *Areg*^fl BM (n=5 mice) and *Areg*^ΔILC2 BM (n=5 mice). **e-g**, Representative AB-PAS-stained images of cecal tips (bars=100 μm) (e). Enumeration of cecal tip goblet cells (f) and crypt length (g) of naïve *Areg*^fl BM (n=4 mice) or *Areg*^ΔILC2 BM (n=3 mice) and *Trichuris*-infected *Areg*^fl BM (n=5 mice) or *Areg*^ΔILC2 BM (n=5 mice). h, Worm burden in *Areg*^fl BM (n=5 mice) or *Areg*^ΔILC2 BM (n=5 mice). **i-l**, Intestinal immune responses in cecum of *Trichuris*-infected *Areg*^fl BM (n=4 mice) and *Areg*^ΔILC2 BM (n=5 mice). Frequencies of ILC2s (i), ST2⁺ Th2 cells (j), and eosinophils (k), and percentages of IL-4⁺ and IL-13⁺ cecal ILC2s and CD4⁺ T cells (l). **m-o,** Gene expression analysis of proximal colons isolated from naïve *Areg*^fl BM (n=4 mice) and *Areg*^ΔILC2 BM (n=3 mice) and *Trichuris*-infected *Areg*^fl BM (n=10 mice) and *Areg*^ΔILC2 BM (n=10 mice). Normalized to *Actb*. Relative expression levels of pro-inflammatory genes (*Il4*, *Il5*, *Il13*, *Ifng*) and immunoregulatory responses (*Il10* and *Tgfb1*) (m). Ratio of type 1 gene (*Ifng*) over type 2 gene (*Il13*) (n). Expression levels of goblet cell functional marker gene *Retnlb* (o). Data in **b-d, f-h, i-l** are representative of two independent experiments. Data in **m-o** are pooled from two independent experiments. Unpaired two-sided t-test (**b-d, h-l**). One-way ANOVA with Tukey multiple comparisons test (**f, g, m-o**). P values are presented where appropriate. ns, not significant. Data are represented as means ± S.E.M.

**Extended Data Figure 7 |. Specific and efficient deletion of *Areg* within T cells in *Areg*^ΔCD4 mice.**
a, WT mice were infected with 200 *Trichuris* eggs by oral gavage, and the cecum was analyzed by flow cytometry for AREG⁺ cells among ILC2s, Foxp3⁺ Tregs and Foxp3⁻ CD4⁺ T effectors at day 0 (n=6 mice), day 3 (n=9 mice), day 9 (n=5 mice), and day 16 (n=4 mice) post-infection. **b-e**, *Areg*^fl (n=6 mice) and *Areg*^ΔCD4 (n=8 mice) were infected with 200 *Trichuris* eggs by oral gavage and analyzed 19 days post-infection. Representative flow cytometry analysis of ILC2s (b) and CD4⁺ T cells (d) for AREG⁺ cells. Percentage of AREG-producing cells within ILC2s (c) and CD4⁺ T cells (e). Data in a are pooled from two independent experiments. Data in c and e are representative of two independent experiments. Unpaired two-sided t-test (**c, e**). P values are presented where appropriate. ns, not significant. Data are represented as means ± S.E.M.

**Extended Data Figure 8 |. Bone marrow chimera mice recapitulated the phenotype of *Areg*^ΔILC2 mice in the context of DSS-induced intestinal damage and inflammation.**
Irradiated CD45.1 WT mice received BM from *Areg*^fl or *Areg*^ΔILC2 donor mice. After 8 weeks, chimera mice were exposed to 3% DSS for 5 days and then allowed to recover for 5 days on regular drinking water. a, Experimental schematic. **b-d**, Examination of AREG deletion in ILC2 (b), ST2⁻ CD4⁺ T cells (c), and ST2⁺ CD4⁺ T cells (d) in DSS-exposed *Areg*^fl BM (n=4 mice) and *Areg*^ΔILC2 BM (n=5 mice) by flow cytometry. **e-g**, Disease severity of DSS-exposed *Areg*^fl BM (n=7 mice) or *Areg*^ΔILC2 BM (n=8 mice) as determined by weight loss (e) and colon length (**f, g**). Data in **b-d** are representative of three independent experiments. Data in e and g are pooled from 2 independent experiments. Unpaired two-sided t-test (**b-d** and g). Two-way ANOVA with Šídák multiple comparisons test (e). P values are presented where appropriate. ns, not significant. Data are represented as means ± S.E.M.

**Extended Data Figure 9 |. Neuronal production of NMU is upregulated in the context of intestinal inflammation.**
a, *Nmu* expression in the proximal colon of naïve (n=4 mice) or *Trichuris*-infected (n=5 mice) determined by qRT-PCR. Normalized to *Actb* housekeeping gene. **b, c** Muscularis propria of naïve water control (n=4 mice) or DSS-exposed inflamed colons (n=4) mice from WT mice were stained for β3-tubulin (white), HuC/D (green), and NMU protein (red). Representative images (bars=50 μm) (b). Neuronal NMU intensity was quantified by normalizing fluorescent intensity of NMU staining to the number of HuC/D⁺ nuclei in two fields per sample (c). d, e, CUBIC imaging of naïve water control (n=3 mice) or DSS-exposed (n=3 mice) colon from WT mice stained for β3-tubulin (green) and NMU protein (red). Representative images (bars=50 μm) (d) and quantification of NMU⁺ pixels (e). f, Representative images of swiss rolls of naïve water or DSS-exposed colons stained for DAPI (white), KLRG1 (cyan), NMU (red) and β3-tubulin (green) (bars=50 μm). Arrows depicting increased NMU staining in DSS-exposed colons. Images are representative of two independent experiments. g, Representative images of swiss rolls of naïve water or DSS-exposed condition colons stained for DAPI (white), Epcam (cyan), NMU (red) and β3-tubulin (green) (bars=50 μm). Images are representative of two independent experiments. Data in a and e are representative of two independent experiments. Data in c are pooled from two independent experiments. Unpaired two-sided t-test (**a, c, e**). P values are presented where appropriate. Data are represented as means ± S.E.M.

**Extended Data Figure 10 |. NMU promotes AREG expression in ILC2s in both mice and humans.**
a, Experimental schematic of RNA sequencing (RNA-seq) analysis of ILC2s and CD4⁺ T cells in the context of intestinal inflammation. Nmur1-eGFP⁺ ILC2s, Nmur1-eGFP⁺ CD4⁺ T cells and Nmur1-eGFP⁻ CD4⁺ T cells were sort-purified from colonic LPLs (n=3 three independent cohorts) and subjected to bulk RNA-seq. b, c, mRNA expression levels of *Nmur1* (b) and *Areg* (c) as determined by comparing the normalized read counts of each gene. d, e, Representative flow cytometry analysis of AREG⁺ CD4⁺ T cells (d) and percentage of AREG⁺ CD4⁺ T cells (e) in the colons of WT mice injected with PBS (n=8 mice) or NMU (n=9 mice). f, Gating strategy for analyzing human colonic LPLs isolated from surgical biopsy samples from ulcerative colitis (UC) patients. **g, h**, AREG production by human CD4⁺ T cells after stimulation of human colonic LPLs from UC patients with or without NMU *in vitro* overnight. Representative flow cytometry analysis of human CD4⁺ T cells (g). Percentage of AREG⁺ CD4⁺ T cells (n=5 donors) (h). Data in **b, c** represent sequenced data from three independent biological replicates. Data in e are pooled from two independent experiments. Data in h contain 5 independent human samples, with each dot representing an individual donor. Two-way ANOVA with Šídák multiple comparisons test (**b, c**). Unpaired two-sided t-test (e). Paired two-sided t-test (h). P values are presented where appropriate. ns, not significant. Data are represented as means ± S.E.M.

**Figure 1 |. Novel mouse strain for efficient targeting of group 2 innate lymphoid cells.**
a, Representative flow cytometry and histogram showing NMUR1 protein expression in colon ILC2s from *Nmur1*^+/+ and *Nmur1*^−/− mice. Total ILCs gated as live CD45⁺Lin⁻CD90⁺CD127⁺ events (Lin: CD3ε, CD5, CD11b, CD11c, FcεRI, B220). b, Representative flow cytometry analysis of NMUR1⁺ cells within colonic lamina propria lymphocytes (LPL) isolated from naïve WT mice as measured by surface protein staining (Lineage 1: CD11b, CD11c, FcεRI, B220. Lineage 2: CD3ε, CD5). **c, d,** Nmur1-eGFP expression in ILCs from Nmur1^iCre-eGFP mice (n=3 mice). Representative overlaid histograms depicting Nmur1-eGFP expression in ILCs (c) and percentages of Nmur1-eGFP⁺ cells within the indicated immune cell subsets isolated from the colon (d). e, Representative overlaid histograms showing iCre (RFP) expression in ILC2s from the indicated tissues of *R26-RFP*^Nmur1 mice. Histograms are representative of two independent experiments. **f, g**, Expression of iCre (RFP) in the indicated progenitor cells isolated from the bone marrow of *R26-RFP*^Nmur1 mice (n=4 mice). Representative histograms (f) and percentages of RFP⁺ cells within indicated progenitor cells (g). h, Representative image of colon from *R26-RFP*^Nmur1 mice (two independent experiments). DAPI (white), CD45 (green), RFP (red) (bar=50 μm). **i, j**, *R26-DTR* (n=11 mice) and *R26-DTR*^Nmur1 (n=7 mice) received 2 daily injections of diphtheria toxin followed by 2 days of rest. Representative flow cytometry analysis of ILC2s in the colon pre-gated on total ILCs (i) and percentages of ILC2s in the indicated tissues (j). Data in d are representative of two independent experiments. Data in g are pooled from two independent experiments. Data in j are pooled from three independent experiments. Two-way ANOVA with Šídák multiple comparisons test (j). P values are presented where appropriate. Data are represented as means ± S.E.M. Neu=neutrophils, Eos=eosinophils, MΦ=macrophages, DC=dendritic cells.

**Figure 2 |. Nmur1^Cre deletes amphiregulin within ILC2s with high efficiency.**
**a, b**, Representative flow cytometry analysis of AREG expression in WT ILC2s (a) and percentages of AREG⁺ ILC2s isolated from SI (n=13 mice), colon (n=13 mice), MLN (n=13 mice), and spleen (n=8 mice) (b). c, d, Representative flow cytometry analysis of AREG⁺ ILC2s (c) and percentages of IL-5⁺ or NMUR1⁺ cells within AREG⁺ ILC2s in MLN isolated from WT mice (n=4 mice) (d). e, f, Representative flow cytometry analysis of ILC2s (e) and percentages of AREG⁺ ILC2s in MLN isolated from *Areg*^fl (n=9 mice), *Areg*^ΔRed5 (n=9 mice) and *Areg*^ΔILC2 (n=7 mice) (f). g-i, Representative flow cytometry analysis of ILC2s (g), percentages of ILC2s within total ILCs (h), and absolute numbers of ILC2s (i) in the colons of *Areg*^fl (n=4 mice) and *Areg*^ΔILC2 (n=5 mice). **j, k**, Representative flow cytometry analysis of IL-5 and IL-13 production in ILC2s (j) and percentages of IL-5⁺IL-13⁺ DP, total IL-5⁺ and total IL-13⁺ cells within ILC2s (k) in the colons of *Areg*^fl (n=4 mice) and *Areg*^ΔILC2 (n=5 mice). Data in b are pooled from three independent experiments. Data in **d, h, i,** and k are representative of two independent experiments. Data from f are pooled from two independent experiments. One-way ANOVA with Tukey multiple comparisons test (b, f). Unpaired two-sided t-test (**h, i,** and k). Paired two-sided t-test (d). P values are presented where appropriate. ns, not significant. Data are represented as means ± S.E.M. DP – double positive.

**Figure 3 |. ILC2-derived AREG is critical for clearance of the gut-dwelling helminth *Trichuris muris* and tissue protection following intestinal injury.**
a, b, Representative flow cytometry analysis of ILC2s (a) and percentages of AREG⁺ ILC2s isolated from WT mice at day 0 (n=12 mice), day 3 (n=9 mice), and day 16 (n=8 mice) post-*Trichuris* infection (b). c, d, Representative flow cytometry analysis of ILC2s (c) and percentages of AREG⁺ ILC2s in the colons of *Areg*^fl (n=9 mice) or *Areg*^ΔILC2 (n=7 mice) (d). e, Total serum IgE levels from naïve *Areg*^fl (n=2 mice) and *Trichuris*-infected *Areg*^fl (n=5 mice) or *Areg*^ΔILC2 (n=5 mice) analyzed at day 19 post-infection. f, g, Goblet cells in the proximal colon of naïve *Areg*^fl (n=3 mice) and *Trichuris*-infected *Areg*^fl (n=5 mice) or *Areg*^ΔILC2 (n=5 mice) analyzed at day 19 post-infection by AB-PAS staining (bars=100 μm) (f). Enumerated goblet cells per crypt (g). **h, i**, *Trichuris* worm counts from *Areg*^fl (n=5 mice) and *Areg*^ΔILC2 (n=5 mice) (h) or *Areg*^fl (n=6 mice) and *Areg*^ΔCD4 (n=8 mice) (i) on day 19 post infection. **j-l**, Measurements of disease severity of DSS-exposed *Areg*^fl (n=16 mice) and *Areg*^ΔILC2 (n=19 mice) as determined by weight loss (j) and colon epithelial crypt architecture analyzed by H&E staining (bars=100 μm) (k, l). Data in b and d are pooled from two independent experiments. Data in **e, g, h** are representative of three independent experiments. Data in i are representative of two independent experiments. Data in j and l are pooled from four independent experiments. One-way ANOVA with Tukey multiple comparisons test (b, e, g). Unpaired two-sided t-test (**d, h, i, l**). Two-way ANOVA with Šídák multiple comparisons test (j). P values are presented where appropriate. ns, not significant. Data are represented as means ± S.E.M.

**Figure 4 |. NMU mediates the non-redundant tissue-protective functions of ILC2-derived AREG.**
a, *Nmu* expression in the naïve (n=4 mice) and DSS-exposed (n=5 mice) distal colons determined by qRT-PCR. Normalized to *Actb* housekeeping gene. b, Representative images of the muscularis propria of naïve (n=3 mice) or DSS-exposed (n=3 mice) inflamed colons of Ai14^Nmu mice (bars=50 μm). β3-tubulin (white), HuC/D (green). **c, d**, Induction of AREG⁺ ILC2s with NMU *in vivo*. Representative flow cytometry analysis of ILC2s (c) and percentages of AREG⁺ ILC2s (d) in the colons of WT mice injected with PBS (n=8 mice) or NMU (n=9 mice) analyzed 18 hours post-injection. **e-h**, Colonic epithelial crypt architecture analyzed by H&E staining (bars=100 μm). DSS-exposed wild-type mice treated with PBS (n=15 mice) or NMU (n=15 mice) (**e, f**). DSS-exposed *Areg*^ΔILC2 mice treated with PBS (n=14 mice) or NMU (n=15 mice) (**g, h**). i, *NMU* expression in the human intestinal biopsies (E-GEOD-14580) from healthy (n=6 donors) or UC patients (n=24 donors) as determined by microarray analysis. j, k, Representative flow cytometry analysis of human ILC2s (j) and percentages of AREG⁺ human ILC2s (k) after stimulation of LPLs isolated from freshly collected surgical colonic and ileal biopsies from IBD patients with or without NMU *in vitro* (n=5 donors). Each dot represents individual donors assessed in independent experiments. Gating strategy is shown in Extended Data Fig. 10f. Data in a are representative of two independent experiments. Data in d are pooled from two independent experiments. Data in f and h are pooled from three independent experiments. Data in k contain 5 independent human samples, with each dot representing an individual donor. Unpaired two-sided t-test (**a, d, f, h, i**). Paired two-sided t-test (k). P values are presented where appropriate. ns, not significant. Data are represented as means ± S.E.M. IBD – inflammatory bowel diseases.

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Pinning down unique ILC2 functions.
Flemming A. Flemming A. Nat Rev Immunol. 2022 Dec;22(12):717. doi: 10.1038/s41577-022-00807-z. Nat Rev Immunol. 2022. PMID: 36357706 No abstract available.

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References

1. Chu C, Artis D & Chiu IM Neuro-immune Interactions in the Tissues. Immunity 52, 464–474 (2020). 10.1016/j.immuni.2020.02.017 - DOI - PMC - PubMed
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[1] Chu C, Artis D & Chiu IM Neuro-immune Interactions in the Tissues. Immunity 52, 464–474 (2020). 10.1016/j.immuni.2020.02.017 - DOI - PMC - PubMed

[2] Chu C, Artis D & Chiu IM Neuro-immune Interactions in the Tissues. Immunity 52, 464–474 (2020). 10.1016/j.immuni.2020.02.017 - DOI - PMC - PubMed

[3] Huh JR & Veiga-Fernandes H Neuroimmune circuits in inter-organ communication. Nat Rev Immunol 20, 217–228 (2020). 10.1038/s41577-019-0247-z - DOI - PubMed

[4] Huh JR & Veiga-Fernandes H Neuroimmune circuits in inter-organ communication. Nat Rev Immunol 20, 217–228 (2020). 10.1038/s41577-019-0247-z - DOI - PubMed

[5] Veiga-Fernandes H & Artis D Neuronal-immune system cross-talk in homeostasis. Science 359, 1465–1466 (2018). 10.1126/science.aap9598 - DOI - PubMed

[6] Veiga-Fernandes H & Artis D Neuronal-immune system cross-talk in homeostasis. Science 359, 1465–1466 (2018). 10.1126/science.aap9598 - DOI - PubMed

[7] Yano H & Artis D Neuronal regulation of innate lymphoid cell responses. Curr Opin Immunol 76, 102205 (2022). 10.1016/j.coi.2022.102205 - DOI - PubMed

[8] Yano H & Artis D Neuronal regulation of innate lymphoid cell responses. Curr Opin Immunol 76, 102205 (2022). 10.1016/j.coi.2022.102205 - DOI - PubMed

[9] Klose CSN et al. The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation. Nature 549, 282–286 (2017). 10.1038/nature23676 - DOI - PMC - PubMed

[10] Klose CSN et al. The neuropeptide neuromedin U stimulates innate lymphoid cells and type 2 inflammation. Nature 549, 282–286 (2017). 10.1038/nature23676 - DOI - PMC - PubMed

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Neuropeptide regulation of non-redundant ILC2 responses at barrier surfaces

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